Seven transmembrane-spanning receptors (7TMRs or G protein-coupled receptors, GPCRs) represent the largest family of signal-transducing molecules known. 7TMRs convey signals for light and many extracellular regulatory molecules, such as, hormones, growth factors and neurotransmitters, that regulate every cell in the body. Dysregulation of 7TMRs has been found in a growing number of human diseases and 7TMRs have been estimated to be the targets of more than 30% of the drugs used in clinical medicine today. Thus, understanding how 7TMRs function is an important goal of biological research. We have used receptors for thyrotropin-releasing hormone (TRH) (TRH-Rs) and for thyroid-stimulating hormone (TSH-R) as model 7TMRs to study their structure and function. During this year, we studied several new aspects of the structure and function of these receptors. 1) An important relatively new observation for several 7TMRs is that they can continue signaling hours after their cognate agonist has been removed. This has been termed persistent signaling. We showed previously that TSH-Rs exhibit persistent signaling. During this year, we showed that persistent signaling by TSH-R is cell autonomous and that in normal, human thyroid cells in primary culture an inhibitor of the phosphodiesterase that degrades cAMP is required to be present to reveal persistent signaling. These findings help explain the different levels of persistent signaling observed in different cell types and show that it occurs in the primary cell in humans that express TSH-Rs. 2) Graves' ophthalmopathy (GO) is a troublesome component of Graves' disease (GD) in 25% of patients for which there is no medical therapy. We study GO in cells taken from the eyes of patients with GO at decompression surgery. We used these cells to study this disease and determined that the most common assay used as an endpoint in these experiments markedly underestimated the cell's response. We established a new assay that accurately measures this endpoint (hyaluronan production). 3) It has recently been shown that TSH acting at the TSH-R can inhibit bone loss in, for example, an animal model of osteoporosis. It was therefore important to begin to determine the signaling pathway within bone cells that mediate this positive effect so as to perhaps develop new approaches to treat osteoporosis. We found, in contrast to the expected signal transduction pathway, that TSH signals through the &#946;-arrestin-1 pathway to stimulated bone development. We are pursuing these observations to develop drug-like molecules to act as probes of this biology and as leads for drug development.